Method for producing acylated peptides

ABSTRACT

The present invention provides a method for acylating one or more amino groups of a peptide where the acylation reaction is to be performed in an aqueous mixture containing less than 10% w/w aprotic polar solvent.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. 119 of Danishapplication no. PA 2002 01421 filed Sep. 25, 2002, and U.S. provisionalapplication No. 60/413,684 filed Sep. 26, 2002, the contents of whichare fully incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for acylating peptidesand proteins. More specifically, the invention relates to a method ofintroducing one or more acyl groups into a peptide or a protein.

BACKGROUND OF THE INVENTION

[0003] A large number of peptides have been approved for use in medicalpractice, and the peptides may be produced in suitable host cells byrecombinant DNA technology or they may be produced synthetically by wellestablished peptide synthesis technology. However, native peptides aswell as analogues thereof tend to exhibit high clearance rates which areunacceptable for many clinical indication where a high plasmaconcentration of the peptide is required over a prolonged period oftime. Examples of peptides which in their native form have a highclearance are: ACTH, corticotropin-releasing factor, angiotensin,calcitonin, insulin, glucagon, glucagon-like peptide-1 (GLP-1),glucagon-like peptide-2 (GLP-2), insulin-like growth factor-1,insulin-like growth factor-2, gastric inhibitory peptide, growthhormonereleasing factor, pituitary adenylate cyclase activating peptide,secretin, enterogastrin, somatostatin, somatotropin, somatomedin,parathyroid hormone, thrombopoietin, erythropoietin, hypothalamicreleasing factors, prolactin, thyroid stimulating hormones, endorphins,enkephalins, vasopressin, oxytocin, opioids and analogues thereof,superoxide dismutase, interferon, asparaginase, arginase, argininedeaminase, adenosine deaminase and ribonuclease.

[0004] A variety of derivatizations of peptides and peptide analogs havebeen found to influence the clearance rate of the peptides in afavourable direction. One such derivatization is the introduction of alipophilic acyl group into the therapeutic peptide causing a desirableprotracted profile of action relative to the non-acylated peptide.Hence, less frequent administration of the therapeutic protein improvesthe patients compliance to the prescribed therapy, and it reduces theamount of peptide to be administered. This has been described anddemonstrated in WO98/08871, which i.a. discloses acylation of GLP-1 andanalogs thereof, in WO98/08872, which i.a. discloses acylation of GLP-2and analogs thereof, and WO99/43708, which i.a. discloses acylation ofexendin and analogs thereof. Mono- or dipeptide spacers such as asparticacid and glutamic acid, between the peptide and the acyl-group wasdemonstrated to be desirable. Spacers including a free carboxylic acidgroup must be protected before acylation and subsequently deprotected.

[0005] EP 1227107 discloses the acylation of ε-amino groups of humaninsulin.

[0006] WO0/55119 discloses a method for acylating peptides (e.g. GLP-1)and novel acylating agents.

[0007] In order for therapeutic peptides to be economically viable thecost of producing the peptides as well as the therapeutic dosage of thepeptide are pivotal. A major cost during production of therapeuticpeptides is the purification steps required to separate the targetprotein from impurities which are closely related to the target protein,e.g. isomers, desamido forms etc. These purification steps are usuallyperformed by chromatography implying expensive chromatography matricesand solvents as well as reduced overall yield.

[0008] It is the aim of the present invention to provide an efficientand economic method for the introduction of lipophilic groups intopeptides via α-amino-α,ω-dicarboxylic acid spacers. The method is morespecific, and thus results in higher yields and reduced formation ofclosely related impurities. A significant reduction of the cost ofproducing the acylated peptides are achieved. Less expensive acylatedpeptides are highly desirable for maximizing the number of patients forwhom the treatment is available as well as for exploiting the advantagesof alternative delivery routes which have lower bioavailability thansubcutaneous injection, e.g. transdermal and pulmonal delivery.

SUMMARY OF THE INVENTION

[0009] The present invention provides a method for acylating one or moreamino groups of a peptide or protein, the method comprising the steps:

[0010] a) reacting the peptide having at least one free amino group withan acylating agent of the general formula I

[0011]  wherein

[0012] n is 0-8;

[0013] R¹ is COOR⁴;

[0014] R² is a lipophilic moiety;

[0015] R³ together with the carboxyl group to which R³ is attacheddesignate a reactive ester or a reactive N-hydroxy imide ester; and

[0016] R⁴ is selected from hydrogen, C₁₋₁₂-alkyl and benzyl,

[0017] under basic conditions in an aqueous mixture;

[0018] b) if R⁴ is not hydrogen, saponifying the acylated peptide estergroup (COOR⁴) under basic conditions;

[0019] c) isolating the N-acylated peptide,

[0020] characterised by said aqueous mixture in step a) containing lessthan 10% w/w aprotic polar solvent.

[0021] In one embodiment of the method, said reaction in step a) takesplace in an aqueous mixture containing less than 8% w/w aprotic polarsolvent, preferably less than 5% w/w aprotic polar solvent and even morepreferable less than 3% w/w aprotic polar solvent.

[0022] In another embodiment of the method, the acylating agent is addedto the reaction mixture as a solid.

[0023] In another embodiment of the method, the acylating agent is addedto the reaction mixture as a solution in a aprotic polar solvent whichis stabilized by adding an acid.

DESCRIPTION OF THE INVENTION

[0024] Peptides and Proteins

[0025] The present invention is useful for the introduction oflipophilic acyl groups into any peptide (or protein) in order to reducethe in vivo clearance rate. Examples of such peptides and proteins areACTH, corticotropin-releasing factor, angiotensin, calcitonin, exendinand analogues thereof, insulin and analogues thereof, glucagon andanalogues thereof, glucagon-like peptide-1 and analogues thereof,glucagon-like peptide-2 and analogues thereof, insulin-like growthfactor-1, insulin-like growth factor-2, gastric inhibitory peptide,growth hormone-releasing factor, pituitary adenylate cyclase activatingpeptide, secretin, enterogastrin, somatostatin, somatotropin,somatomedin, parathyroid hormone, thrombopoietin, erythropoietin,hypothalamic releasing factors, prolactin, thyroid stimulating hormones,endorphins, enkephalins, vasopressin, oxytocin, opiods and analoguesthereof, superoxide dismutase, interferon, asparaginase, arginase,arginine deaminase, adenosine deaminase and ribonuclease.

[0026] It should be understood that the peptide (or protein) shouldcarry at least one free amino group, such an amino group being theN-terminal amino group or a side chain amino group. The peptides orprotein may comprise amino acids which are not encoded by the geneticcode, such as D-amino acids, 3-hydroxyproline, ornithine andpentylglycine. Particularly interesting are amino groups of lysine andornithine amino acid residues. The method is particular relevant for theN-acylation of the ε-amino group of lysine residues. It should also beunderstood that the peptide or protein in question may comprise two ormore pendant amino groups which all may be N-acylated according to thepresent invention.

[0027] The present invention is especially suitable for the acylation ofGLP-1 and analogues thereof. Examples of GLP-1 and analogues which canbe N-acylated according to the present invention are GLP-1 and truncatedanalogues, such as Arg²⁶-GLP-1(7-37); Arg³⁴-GLP-1(7-37);Lys³⁶-GLP-1(7-37); Arg^(26,34)Lys³⁶-GLP-1(7-37);Arg^(26,34)Lys³⁹GLP-1(7-38); Arg^(26.34)Lys³⁹-GLP-1(7-39);Arg^(26,34)Lys⁴⁰-GLP-1(740); Arg³⁴Lys³⁶-GLP-1(7-37);Arg²⁶Lys³⁹-GLP-1(7-39); Arg³⁴Lys⁴⁰-GLP-1(7-40);Arg^(26,34)Lys^(36,39)-GLP-1(7-39); Arg^(26,34)Lys^(36,40)-GLP-1(7-40);Gly⁸Arg²⁶-GLP-1(7-37); Gly⁸Arg³⁴-GLP-1(7-37); Gly⁸Lys³⁶-GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶-GLP-1(7-37); Gly⁸Arg^(26,34)Lys³⁹-GLP-1(7-39);Gly⁸Arg^(26,34)Lys⁴-GLP-1(7-40); Gly⁸Arg²⁶Lys³⁶-GLP-1(7-37);Gly⁸Arg³⁴Lys³⁶-GLP-1(7-37); Lys³⁶-GLP-1(7-37);Arg^(26,34)Lys³⁶-GLP-1(7-37); Arg^(26,34)-GLP-1(7-37);Arg^(26,34)Lys⁴⁰-GLP-1(7-37); Arg²⁶Lys³⁶-GLP-1(7-37);Arg³⁴Lys³⁶-GLP-1(7-37); Val⁸Arg²²-GLP-1(7-37); Met⁸Arg²²-GLP-1(7-37);Gly⁸His²²-GLP-1(7-37); Val⁸His²²-GLP-1(7-37); Met⁸His²²-GLP-1(7-37);His³⁷-GLP-1(7-37); Gly⁸-GLP-1(7-37); Val⁸-GLP-1(7-37); Met⁸-GLP-1(7-37);Gly⁸Asp²²-GLP-1(7-37); Val⁸Asp²²-GLP-1(7-37); Met⁸Asp²²-GLP-1(7-37);Gly⁸Glu²²-GLP-1(7-37); Val⁸ Glu²²-GLP-1(7-37); Met⁸Glu²²-GLP-1(7-37);Gly⁸Lys²²-GLP-1(7-37); Val⁸Lys²²-GLP-1 (7-37); Met⁸Lys²²-GLP-1(7-37);Gly⁸Arg²²-GLP-1(7-37); Val⁸Lys²²His³⁷-GLP-1(7-37);Gly⁸Glu²²His³⁷-GLP-1(7-37); Val⁸Glu²²His³⁷-GLP-1(7-37);Met⁸Glu²²His³⁷-GLP-1(7-37); Gly⁸Lys²² His³⁷-GLP-1(7-37);Met⁸Lys²²His³⁷-GLP-1(7-37); Gly⁸Arg²²His³⁷-GLP-1(7-37);Val⁸Arg²²His³⁷-GLP-1(7-37); Met⁸Arg²²His³⁷-GLP-1(7-37);Gly⁸His²²His³⁷-GLP-1(7-37); Val⁸His²²His³⁷-GLP-1(7-37);Met⁸His²²His³⁷-GLP-1(7-37); Gly⁸His³⁷-GLP-1(7-37);Val⁸His³⁷-GLP-1(7-37); Met⁸His³⁷-GLP-1(7-37); Gly⁸Asp²²His³⁷-GLP-1(7-37); Val⁸Asp²²His³⁷-GLP-1(7-37);Met⁸Asp²²His³⁷-GLP-1(7-37); Arg²⁶-GLP-1(7-36)-amide;Arg³⁴-GLP-1(7-36)-amide; Lys³⁶-GLP-1(7-36)-amide;Arg^(26,34)Lys³⁶-GLP-1(7-36)-amide; Arg^(26,34)-GLP-1(7-36)-amide;Arg^(26,34)Lys⁴⁰-GLP-1(7-36)-amide; Arg²⁶Lys³⁶-GLP-1(7-36)-amide;Arg³⁴Lys³⁶-GLP-1(7-36)-amide; Gly⁸-GLP-1(7-36)-amide;Val⁸-GLP-1(7-36)-amide; Met⁸-GLP-1(7-36)-amide;Gly⁸Asp²²-GLP-1(7-36)-amide; Gly⁸Glu²²His³⁷-GLP-1(7-36)-amide;Val⁸Asp²²-GLP-1(7-36)-amide; Met⁸Asp²²-GLP-1(7-36)-amide;Gly⁸Glu²²-GLP-1(7-36)-amide; Val⁸Glu²²-GLP-1(7-36)-amide;Met⁸Glu²²-GLP-1(7-36)-amide; Gly⁸Lys²²-GLP-1(7-36)-amide;Val⁸Lys²²-GLP-1(7-36)-amide; Met⁸Lys²²-GLP-1(7-36)-amide;Gly⁸His²²His³⁷-GLP-1(7-36)-amide; Gly⁸Arg²²-GLP-1(7-36)-amide;Val⁸Arg²²-GLP-1(7-36)-amide; Met⁸Arg²²-GLP-1(7-36)-amide;Gly⁸His²²-GLP-1(7-36)-amide; Val⁸His²²-GLP-1(7-36)-amide;Met⁸His²²-GLP-1(7-36)-amide; His³⁷-GLP-1(7-36)-amide;Val⁸Arg²²His³⁷-GLP-1(7-36)-amide; Met⁸Arg²²His³⁷-GLP-1(7-36)-amide;Gly⁸His³⁷-GLP-1(7-36)-amide; Val⁸His³⁷-GLP-1(7-36)-amide;Met⁸His³⁷-GLP-1(7-36)-amide; Gly⁸Asp²² His³⁷-GLP-1(7-36)-amide;Val⁸Asp²²His³⁷-GLP-1(7-36)-amide; Met⁸Asp²²His³⁷-GLP-1(7-36)-amide;Val⁸Glu²²His³⁷-GLP-1(7-36)-amide; Met⁸Glu²²His³⁷-GLP-1 (7-36)-amide;Gly⁸Lys²² His³⁷-GLP-1(7-36)-amide; Val⁸Lys²²His³⁷-GLP-1(7-36)-amide;Met⁸Lys²²His³⁷-GLP-1(7-36)-amide; Gly⁸Arg²²His³⁷-GLP-1(7-36)-amide;Val⁸His²²His³⁷-GLP-1(7-36)-amide; Met⁸His²²His³⁷-GLP-1(7-36)-amide; andderivatives thereof.

[0028] Each of these GLP-1 analogues and truncated analogues constitutesalternative embodiments of the present invention.

[0029] The present invention is also especially suitable for theacylation of GLP-2 and analogues thereof. Examples of GLP-2 andanalogues which can be N-acylated according to the present invention areGLP-2 analogues and truncated analogues, such as Lys²⁰GLP-2(1-33);Lys²⁰Arg³⁰GLP-2(1-33); Arg³⁰Lys³⁴GLP-2(1-34); Arg³⁰Lys³⁵GLP-2(1-35);Arg^(30,35)Lys²⁰GLP-2(1-35); and Arg³⁵GLP-2(1-35). Each of these GLP-2analogues and truncated analogues constitutes alternative embodiments ofthe present invention.

[0030] The present invention is also especially suitable for theacylation of exendin-3 and exendin-4 and analogues thereof. Examples ofexendin analogues which can be N-acylated according to the presentinvention are disclosed in e.g. WO99/43708. Each of these exendinanalogues and truncated analogues constitutes alternative embodiments ofthe present invention.

[0031] The present invention is also particularly suited for theacylation of insulin and analogues thereof. Examples of insulin andanalogues thereof which can be N-acylated according to the presentinvention are human insulin and des(B30)-human insulin.

[0032] In a further embodiment of the present invention the N-acylationtakes place at the ε-amino group of lysine residues.

[0033] Acylating Agent

[0034] In the method according to the invention, a peptide (or protein)which has at least one free amino group is reacted with an acylatingagent of the general formula I

[0035] The integer n in formula I is preferably 0-8, in particular 0-6corresponding, e.g., to aspartic acid, glutamic acid, etc. Preferably, nis 0-4 such as 0-2, e.g. 0 (aspartic acid) or 1 (glutamic acid). Each ofthese integers and ranges constitutes alternative embodiments of thepresent invention.

[0036] R¹ in formula I represents a free acid group (COOH) or an estergroup (COOR⁴). In the cases where R¹ is an ester group, R⁴ is selectedfrom groups which can be removed (as the corresponding alcohols) byhydrolysis under basic conditions. Examples of such groups areC₁₋₁₂-alkyl, e.g. methyl, ethyl, prop-1-yl, prop-2-yl, but-1-yl,but-2-yl, 2-methyl-prop-1-yl, 2-methyl-prop-2-yl (tert-butyl), hex-1-yl,etc., and benzyl. Each of these groups constitutes alternativeembodiments of the present invention.

[0037] R² in formula I represents the lipophilic moiety to beincorporated into the peptide or protein. Such a lipophilic moiety istypically selected from C₃₋₃₉-alkyl, C₃₋₃₉-alkenyl, C₃₋₃₉-alkadienyl andsteroidal residues. Specific examples of C₃₋₃₉-alkyl are heptyl, nonyl,undecanyl, tridecanyl, pentadecanyl, heptadecanyl, and nonadecanyl. Eachof these lipophilic moieties constitutes alternative embodiments of thepresent invention.

[0038] The lipophilic substituent or moiety is characterised by having asolubility in water at 20° C. in the range from about 0.1 mg/100 mlwater to about 250 mg/100 ml water, preferable in the range from about0.3 mg/100 ml water to about 75 mg/100 ml water. For instance, octanoicacid (C8) has a solubility in water at 20° C. of 68 mg/100 ml, decanoicacid (C10) has a solubility in water at 20° C. of 15 mg/100 ml, andoctadecanoic acid (C18) has a solubility in water at 20° C. of 0.3mg/100 ml. Each of these lipophilic substituent ranges constitutesalternative embodiments of the present invention.

[0039] The terms “C₃₋₃₉-alkyl”, “C₃₋₃₉-alkenyl” and “C₃₋₃₉-alkadienyl”is intended to cover straight chain and branched, preferably straightchain, saturated, mono-unsaturated and diunsaturated, respectively,hydrocarbon radicals of 3-39 carbon atoms. Specific examples ofC₃₋₃₉-alkyl are heptyl, nonyl, undecanyl, tridecanyl, pentadecanyl,heptadecanyl, and nonadecanyl.

[0040] When used herein, the term “steroidal residue” is intended tomean a lipophilic group which together with the carbonyl group to whichR² is attached is derived from a steroide carboxylic acid, i.e. a tri-,tetra- and pentacyclic, full saturated or partially unsaturatedC₁₆₋₃₆-hydrocarbon. Examples of such groups R²—C(═O)— are lithocholoyl,deoxycholoyl, and choloyl.

[0041] Among the lipophilic groups mentioned above, C₇₋₂₅-alkyl,C₇₋₂₅-alkenyl, C₇₋₂₅-alkadienyl and steroidal residues are especiallyrelevant. Particularly interesting examples are heptyl, nonyl,undecanyl, tridecanyl, pentadecanyl, heptadecanyl, nonadecanyl,lithocholoyl, deoxycholoyl, and choloyl. Each of these lipophilic groupsconstitutes alternative embodiments of the present invention.

[0042] R³ in formula I together with the carboxyl group to which R³ isattached designate a reactive ester or a reactive N-hydroxy imide ester.Each of these esters constitutes alternative embodiments of the presentinvention. Reactive esters and reactive N-hydroxy imide esters are wellknown in the art of organic chemistry (especially in peptide chemistry)as functional groups which are used in acylation of amino, thio andhydroxy groups. Within the context of the present invention, the term“reactive ester or a reactive N-hydroxy imide ester” is intended to meanan ester functionalised form of a carboxylic acid group suitable foracylating an amine, preferably an primary amine. It should, thus, beunderstood, that selectivity for acylation of primary amines ispreferred over acylating of hydroxy and thio groups. Reactive N-hydroxyimide esters are especially preferred.

[0043] Examples of reactive esters are 1-hydroxybenzotriazole esters andderivatives. A number of highly effective reagents, e.g.2-(1H-benzotriazol-1yl)-1,1,3,3,-tetramethyluronium tetrafluoroborate,for the formation of such activated esters of carboxylic acids areknown. Such reactive esters are typically formed in situ in the presenceof a base, e.g. an organic base such as an trialkylamine.

[0044] Examples of the imide part of reactive N-hydroxy imide esters arethose specifically described in EP 0511600 A2, page 3 to page 7.Especially interesting examples of imide parts among those aresuccinimide, phthalimide, etc. Each of these imide parts constitutesalternative embodiments of the present invention.

[0045] The reactive N-hydroxy imide esters of the formula I can beprepared as described in WO0/55119 and WO98/02460.

[0046] In the event where the acylating reagent of the formula I is tobe used as the free α-carboxylic acid (R⁴=hydrogen), a compound of theformula I where R⁴ is a group which can be removed selectively isconverted to the corresponding compound where R⁴ is hydrogen. Thecarboxylic acid protecting group may be a benzyl group which can beremoved by catalytic hydrogenation or an allyl group which can beselectively removed. A benzyl protecting group may be removed bycatalytic hydrogenation in an aprotic polar solvent, e.g. in acetone, atroom temperature by using palladium-on-carbon and hydrogen. The reactionmay be performed in a closed vessel with an hydrogen atmosphere(typically 0.1-10 atm) under vigorous stirring. The reaction istypically completed within 0.5-12 hours depending on the quality of thepalladium catalyst. Conventional work-up applies.

[0047] R Action Conditions

[0048] The reaction between the acylating agent of the formula I and thepeptide or protein is performed under basic conditions in an aqueoussolution containing less than 10% w/w of an aprotic polar solvent.

[0049] In one embodiment of the invention the reaction in step a) isperformed in an aqueous solution containing from 0% w/w to 10% w/w of anaprotic polar solvent.

[0050] In another embodiment of the invention the reaction in step a) isperformed in an aqueous solution containing from 1% w/w to 10% w/w of anaprotic polar solvent.

[0051] In another embodiment of the invention the reaction in step a) isperformed in an aqueous solution containing from 1% w/w to 8% w/w of anaprotic polar solvent.

[0052] The acylating agent of the formula I is typically used in aslight excess relative to the number of amino groups of the peptide tobe acylated. The ratio is typically 1:1 to 1:20 with an excess of theacylating agent, preferably 1:1.2 to 1:5, taking into account the numberof amino groups in the peptide. The acylating agent may be added to thereaction mixture as a solid or it may be added to the reaction mixtureas a solution. When the acylating agent is added as a solution it isdissolved in an aprotic polar solvent and preferably stabilized byadding an acid. Typically, the acid for the stabilization is a mineralacid, e.g. sulfuric acid.

[0053] It should be understood that the peptide may be fully N-acylatedor only partially N-acylated depending on the amount of acylating agentused and the reaction conditions. It is preferred that the N-acylationis substantially stoichiometrical.

[0054] Aprotic polar solvents are solvents with moderately highdielectric constants which do not contain acidic hydrogen (see e.g.Morrison and Boyd, Organic Chemistry, 5^(th) ed. p 229). Typically, theaprotic polar solvent is selected from anhydrous tetrahydrofuran (THF),anhydrous dimethylformamide (DMF), acetone, dichloromethane,dimethylsulfoxide (DMSO), dioxane, dimethylacetamide, andN-methyl-2-pyrrolidone and mixtures thereof, among whichdimethylformamide, dimethylsulfoxide, dimethylacetamide andN-methyl-2-pyrrolidone are preferred and N-methyl-2-pyrrolidone isespecially preferred.

[0055] The temperature is typically kept in the range of −10-50° C.

[0056] It is important that the pH value of the solvent mixture is inthe range of 7-14, such as 9-13, preferably in the range of 10-12, inorder for the reaction to proceed smoothly. The result with respect toyield and purity is normally optimal when the pH value of the solventmixture is in the range of 10-12. The desired pH value is obtained byaddition of alkalimetal hydroxides, e.g. sodium hydroxide and potassiumhydroxide, and/or organic bases such as trialkylamines (e.g.triethylamine, N,N-diisopropylethylamine, etc.). It may also beadvantageous to add a buffer which is suitable for keeping the pH nearthe starting value before the reaction starts. Examples of buffer whichmay be used for this purpose is phosphate buffer, borate buffer and thelike.

[0057] As a typical example, the reaction in step (a) is performed usingthe protein and the acylating agent of the formula I in a 1:1 to 1:5molar ratio. The peptide is typically pre-dissolved in water at −10-30°C. such as 0-25° C. and the pH is adjusted to the desired level using aalkalimetal hydroxide (e.g. sodium hydroxide or potassium hydroxide).The pH value may be further adjusted using acids, e.g. acetic acid, andbases, e.g. trialkylamine, but the temperature is preferably within theabove range. Alternatively the peptide is pre-dissolved directly in anaqueous solution of an appropriate amount of the relevant acid or base.The acylating agent is subsequently added as a solid or as a solution inan aprotic polar solvent. The reaction is typically allowed to proceedto completion (can be monitored by HPLC) which is typically obtainedwithin 0.2-4 hours, such as 0.2-1 hour, before addition of water and anacid, e.g. acetic acid, to pH 6.5-9.0. The product is typically isolatedand purified by HPLC, or is precipitated by isoelectric pH, or ishydrolysed (step (b)) before purification.

[0058] When an acylating agent of the formula I where R⁴ is hydrogen isused, the N-acylated peptide or protein carrying lipophilic moieties andfree carboxylic groups is obtained directly. Thus, the variant where R⁴is hydrogen represents a preferred embodiment of the method of thepresent invention.

[0059] Alternatively, i.e. when the group R⁴ is C₁₋₁₂-alkyl or benzyl,the N-acylated peptide ester (or protein ester) is saponified underbasic conditions so as to obtain the N-acylated peptide or N-acylatedprotein. Saponification is typically performed in a 0.01-4.0 M solutionof an alkali-metal hydroxide, e.g. sodium or potassium hydroxide. The pHof the solution is typically 10-14. The reaction is typically allowed toproceed for 0.1-12 hours, preferably for 0.5-4 hours, at 0-40° C. suchas around room temperature. After reaction, the product is purified,e.g. by precipitation at isoelectric pH and/or by preparative HPLC.Thus, the variant where R⁴ is C₁₋₁₂-alkyl or benzyl represents anotherpreferred embodiment of the method of the present invention.

[0060] In one embodiment of the method, the acylating agent is added tothe reaction mixture as a solid.

[0061] In another embodiment of the method, said reaction in step a)takes place in an aqueous mixture containing from 0% w/w to 8% w/waprotic polar solvent, preferably from 0% w/w to 5% w/w aprotic polarsolvent and even more preferable from 0% w/w to 3% w/w aprotic polarsolvent.

[0062] In yet another embodiment of the method, said reaction in step a)takes place in the presence of an aprotic polar solvent, and saidaprotic polar solvent is selected from the group consisting ofN-methyl-2-pyrrolidone, tetrahydrofurane and dimethylsulfoxide.

[0063] In yet another embodiment of the method, all of the aproticorganic solvent is added to the reaction mixture as a solvent for theacylating agent.

[0064] In yet another embodiment of the method, the acylating agent isadded to the reaction mixture as a solution which is stabilized byadding an acid.

[0065] In yet another embodiment of the method, said acid is added tothe aprotic polar solvent in a concentration from 0.01% w/w to 1% w/w,preferably in a concentration from 0.05% w/w to 0.5% w/w.

[0066] In yet another embodiment of the method, said acid is selectedfrom the group consisting of sulphuric acid, methanesulphonic acid andtrifluoroacetic acid.

[0067] In yet another embodiment of the method, the reaction in step a)takes place in the absence of an aprotic polar solvent.

[0068] In yet another embodiment of the method, R⁴ in formula I ishydrogen.

[0069] In yet another embodiment of the method, R⁴ is selected fromC₁₋₈-alkyl and benzyl.

[0070] In yet another embodiment of the method, R³ together with thecarboxyl group to which R³ is attached designate a reactive N-hydroxyimide ester.

[0071] In yet another embodiment of the method, the acylated peptideester is saponified at a pH value in the range of 10-14, preferably inthe pH range from 9-13.

[0072] In yet another embodiment of the method, the acylated peptideester is saponified at a pH value in the range of 9-14, such as in thepH range from 10-13.

[0073] In yet another embodiment of the method, pH of the reactionmixture in step a) is from pH 9 to pH 13, preferably from pH 10 to pH12, or more preferable from pH 11.0 to pH 11.5.

[0074] In yet another embodiment of the method, the reaction mixture instep a) comprises a buffer which is suitable for maintaining asubstantially constant pH during the reaction. In one embodiment of themethod said buffer is a phosphate buffer, a borate buffer or a mixturethereof.

[0075] In yet another embodiment of the method, the temperature of thereaction mixture in step a) is in the range of 0-50° C., preferably inthe range from 5-40° C. and more preferable in the range from 10-30° C.

[0076] In yet another embodiment of the method, R² is selected fromC₃₋₃₉-alkyl, C₃₋₃₉-alkenyl, C₃₋₃₉-alkadienyl and steroidal residues.

[0077] In yet another embodiment of the method, R²—C(═O)— is selectedfrom the group consisting of lithocholoyl and hexadecanoyl.

[0078] In yet another embodiment of the method, said peptide used asstarting material for step a) has a peptide purity of at least 80%, atleast 90%, at least 93%, at least 95%, or at least 97% as determined byRP-HPLC.

[0079] In yet another embodiment of the method, said peptide is selectedfrom the group consisting of GLP-1, exendin-4, GLP-2, glucagon, insulin,analogues thereof and derivatives of any of the foregoing.

[0080] In yet another embodiment of the method, said peptide is a GLP-1agonist.

[0081] In yet another embodiment of the method, said peptide is selectedfrom the group consisting of exendin-3, exendin-4, Arg³⁴-GLP-1(7-37),Gly⁸-GLP-1(7-36)-amide, Gly⁸-GLP-1(7-37), Val⁸-GLP-1(7-36)-amide,Val⁸-GLP-1(7-37), Val⁸Asp²²-GLP-1(7-36)-amide, Val⁸Asp²²-GLP-1(7-37),Val⁸Glu²²-GLP-1(7-36)-amide, Val⁸Glu²²-GLP-1(7-37),Val⁸Lys²²-GLP-1(7-36)-amide, Val⁸Lys²²-GLP-1(7-37),Val⁸Arg²²-GLP-1(7-36)-amide, Val⁸Arg²²-GLP-1(7-37),Val⁸His²²-GLP-1(7-36)-amide, Val⁸His²²-GLP-1(7-37), des(B30) humaninsulin and derivatives thereof.

[0082] In yet another embodiment of the method, said peptide is selectedfrom the group consisting of consisting of Val⁸Trp¹⁹GU²²-GLP-1(7-37),Val⁸Glu²²Val²²-GLP-1(7-37), Val⁸Tyr¹⁶Glu²²-GLP-1(7-37),Val⁸Trp¹⁶Glu²²-GLP-1(7-37), Val⁸Leu¹⁶Glu²²-GLP-1(7-37),Val⁸Tyr¹⁸Glu²²-GLP-1(7-37), Val⁸Glu²² His³⁷-GLP-1(7-37),Val⁸Glu²²Ile³³-GLP-1(7-37), Val⁸Trp¹⁶Glu²²Val²⁵Ile³³-GLP-1(7-37),Val⁸Trp¹⁶Glu²²Ile³³-GLP-1(7-37), Val⁸Glu²²Val²⁵Ile³³-GLP-1(7-37),Val⁸Trp¹⁶Glu²²Val²⁵-GLP-1(7-37) and analogues thereof.

[0083] In yet another embodiment of the method, said peptide is selectedfrom HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2 (ZP-10) andanalogues thereof.

[0084] In another embodiment of the method, said peptide is not aninsulin peptide, i.e. it is not insulin or an analogue thereof. In yetanother embodiment of the method, said peptide comprises only onepolypeptide chains. In yet another embodiment of the method, saidpeptide comprises two polypeptide chains which are covalently connectedby at least one disufide bond.

[0085] Another aspect of the present invention is the use of theacylation method for the preparation of a peptide derivative selectedfrom the group consisting of Arg³⁴, Lys²⁶(N^(ε-(γ-Glu(N)^(α)-hexadecanoyl)))-GLP-1(7-37) and LysB²⁹(N^(ε)-tetradecanoyl)des(B30) human insulin and LyS^(B29)(N^(ε)[N^(α)-lithocholoyl-Glu-OH])des(B30) human insulin.

EXAMPLES

[0086] Preparation of the Acylation Reagents is Done as Described in WO00/55119.

[0087] In the examples final purification of the product was obtained bycolumn chromatography.

Example 1

[0088] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed in yeast (S. cerevisiae) byconventional recombinant DNA technology, e.g. as described in WO98/08871. Arg³⁴GLP-1₍₇₋₃₇₎ in the fermentation broth was then purifiedby conventional reversed phase chromatography and subsequentlyprecipitated at the isoelectric pH of the peptide, i.e. at pH 5.4. Theprecipitate was isolated by centrifugation and frozen.

[0089] Arg³⁴-GLP-1⁷⁻³⁷ (1.47 g of frozen iso-precipitated peptidematerial, approx. 0.10 mmol) was dissolved in 0.1 mol/kg triethylamine(23 ml) at 10-15° C. The pH of the solution was 11.6.N-hexadecanoylglutamic acid γ-N-hydroxysuccinimide ester (63.7 mg, 0.13mmol) was added. After 20 minutes at room temperature water (42 ml) wasadded, and the pH was adjusted to 8.0 by addition of 1.0 M acetic acid.

[0090] Yield: By analytical RP-HPLC the reaction mixture was shown tocontain 84% (by area) ofArg³⁴Lys²⁶-[N-ε-(ε-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷ and 0.5% (by area) ofArg³⁴Lys²⁶-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷.

Example 2

[0091] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed in yeast (S. cerevisiae) byconventional recombinant DNA technology, e.g. as described in WO98/08871. Arg³⁴GLP-1₍₇₋₃₇₎ in the fermentation broth was then purifiedby conventional reversed phase chromatography and subsequentlyprecipitated at the isoelectric pH of the peptide, i.e. at pH 5.4. Theprecipitate was isolated by centrifugation and frozen.

[0092] Arg³⁴-GLP-1⁷⁻³⁷ (1.57 g of frozen iso-precipitated peptidematerial, approx. 0.14 mmol) was dissolved in 0.1 mol/kg triethylamine(23 ml) at 10-15° C. The pH of the solution was adjusted to 11.5 by theaddition of triethylamine. 1.7 mL of N-hexadecanoylglutamic acidγ-N-hydroxysuccinimide ester (92.1 mg, 0.19 mmol) dissolved inN-methyl-2-pyrrolidone containing 0.105% w/w 1 M H₂SO₄ was added. After20 minutes at room temperature water (42 ml) was added, and the pH wasadjusted to 8.0 by addition of 1.0 M acetic acid.

[0093] Yield: By analytical RP-HPLC the reaction mixture was shown tocontain 83% (by area) ofArg³⁴Lys²⁶-[N-ε-(γ-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷ and 0.4% (by area) ofArg³⁴Lys²⁶-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-1^(7-37.)

Example 3

[0094] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed in yeast (S. cerevisiae) byconventional recombinant DNA technology, e.g. as described in WO98/08871. Arg³⁴GLP-1₍₇₋₃₇₎ in the fermentation broth was then purifiedby conventional reversed phase chromatography and subsequentlyprecipitated at the isoelectric pH of the peptide, i.e. at pH 5.4. Theprecipitate was isolated by centrifugation and frozen.

[0095] Arg³⁴-GLP-1⁷⁻³⁷ (1.53 g of frozen iso-precipitated peptidematerial, approx. 0.13 mmol) was dissolved in 0.05 mol/kg triethylamine(25 ml) at room temperature. The pH of the solution was 10.9.1.8 ml ofN-hexadecanoylglutamic acid γ-N-hydroxysuccinimide ester (94.2 mg, 0.19mmol) dissolved in N-methyl-2-pyrrolidone without H₂SO₄ was added. After30 minutes at room temperature water (48 ml) was added, and the pH wasadjusted to 8.0 by addition of 1.0 M acetic acid.

[0096] Yield: By analytical RP-HPLC the reaction mixture was shown tocontain 75% (by area) ofArg³⁴Lys²⁶-[N-ε-(γ-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷ and 4.0% (by area) ofArg³⁴Lys²⁶-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷.

Example 4 Reference Example with Aprotic Polar Solvent conc. >10% w/w.

[0097] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed in yeast (S. cerevisiae) byconventional recombinant DNA technology, e.g. as described in WO98/08871. Arg³⁴GLP-1₍₇₋₃₇₎ in the fermentation broth was then purifiedby conventional reversed phase chromatography and subsequentlyprecipitated at the isoelectric pH of the peptide, i.e. at pH 5.4. Theprecipitate was isolated by centrifugation and frozen.

[0098] Arg³⁴-GLP-1⁷⁻³⁷ (1.57 g of frozen iso-precipitated peptidematerial, approx. 0.14 mmol) was dissolved in 0.1 mol/kg triethylamine(23 ml) at 10-15° C. N-methyl-2-pyrrolidone (6.8 ml) was added and thepH of the solution was adjusted to 11.5 by the addition oftriethylamine. Then N-hexadecanoylglutamic acid γ-N-hydroxysuccinimideester (92.1 mg, 0.19 mmol) dissolved in 1.7 ml N-methyl-2-pyrrolidonecontaining 0.105% w/w 1 M H₂SO₄ was added. After 20 minutes at roomtemperature water (54 ml) was added, and the pH was adjusted to 8.0 byaddition of 1.0 M acetic acid.

[0099] Yield: By analytical RP-HPLC the reaction mixture was shown tocontain 87% (by area) ofArg³⁴Lys²⁶-[N-ε-(γ-Glu(N-hexadecanoyl))]-GLP-17-37 and 0.5% (by area) ofArg³⁴Lys²⁶-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷.

Example 5 Reference Example with Aprotic Polar Solvent conc. >10% w/w.

[0100] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed in yeast (S. cerevisiae) byconventional recombinant DNA technology, e.g. as described in WO98/08871. Arg³⁴GLP-1₍₇₋₃₇₎ in the fermentation broth was then purifiedby conventional reversed phase chromatography and subsequentlyprecipitated at the isoelectric pH of the peptide, i.e. at pH 5.4. Theprecipitate was isolated by centrifugation and frozen.

[0101] Arg³⁴-GLP-1⁷⁻³⁷ (1.51 g of frozen iso-precipitated peptidematerial, approx. 0.13 mmol) was dissolved in 0.1 mol/kg triethylamine(20 ml) and N-methyl-2-pyrrolidone (100 ml) at 10-15° C. ThenN-hexadecanoylglutamic acid γ-N-hydroxysuccinimide ester (74 mg, 0.15mmol) dissolved in 1.4 ml N-methyl-2-pyrrolidone containing 0.105% w/w 1M H₂SO₄ was added. After 45 minutes at room temperature water (206 ml)was added, and the pH was adjusted to 8 by addition of 1.0 M aceticacid.

[0102] Yield: By analytical RP-HPLC the reaction mixture was shown tocontain 57% (by area) ofArg³⁴Lys²⁶-[N-ε-(γ-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷ and 14% (by area) ofArg³⁴Lys²⁶-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷.

Example 6

[0103] In table 1 the experimental conditions used for acylatingArg³⁴-GLP-1⁷⁻³⁷ in the experiments of example 1-5 are summarised and theresulting contents of the impurityArg³⁴Lys²⁶-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷ (abbreviated α-glu)are listed. Without the addition of H₂SO₄ as stabilizer it is clear thatlower concentrations of aprotic polar solvent is effective in reducingthe amount of the α-glu impurity. When the stabilizer is added theformation of significant amounts of the α-glu impurity start atsignificantly higher concentrations of aprotic polar solvent, i.e. abovethe concentration 28% w/w. TABLE 1 The contents of aprotic polar solventin the reaction mixture when acylating GLP-1 peptides and the resultingfraction of the impurityArg³⁴Lys²⁶-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷ (α-glu). (Data fromExamples 1-5). Aqueous peptide NMP NMP con- Example solution added tentsH₂SO₄ # (mL) (mL) (% w/w) added α-glu 1 23 0 0% − 0.5% 2 23 1.7 7% +0.4% 3 25 1.8 7% −   4% 4 23 6.8 + 1.7 28%  + 0.5% 5 20 100 + 1.4 84%  + 14%

Exampl 7

[0104] Sodium-crystallized des(B30)-human insulin (1.74 g of frozenpeptide material isolated by isoelectric precipitation, approx. 0.088mmol) was dissolved in 0.1 mol/kg triethylamine (10.85 ml) at roomtemperature. The pH of the solution was 10.9. Then, methyl(2S)-5-[(2,5-dioxo-1-pyrrolidinyl)oxy]-2-[(3a,5b)-3-hydroxy-9-methyl-24-oxocholan-24-yl]amino-5-oxopentanoate (55.1mg, 0.089 mmol) dissolved in N-methyl-2-pyrrolidone (1.25 ml) was added.After 30 minutes at room temperature water (5° C., 85 ml) was added.

[0105] Yield: By analytical RP-HPLC the reaction mixture was shown tocontain 34% (by area) ofNε-(lithocholoyl-γ-glutamyl)-LyS^(B29)-des(B30)-human insulin.

Example 8

[0106] Arg³⁴GLP-1₍₇₋₃₇₎ was expressed in yeast (S. cerevisiae) byconventional recombinant DNA technology, e.g. as described in WO98/08871. Arg³⁴GLP-1₍₇₋₃₇₎ in the fermentation broth was then purifiedby conventional reversed phase chromatography and subsequentlyprecipitated at the isoelectric pH of the peptide, i.e. at pH 5.4. Theprecipitate was isolated by centrifugation and frozen.

[0107] The isoelectric precipitate containing Arg³⁴GLP-1₍₇₋₃₇₎ (5.70 gof frozen precipitated peptide material, approx. 0.38 mmol) wasdissolved in 0.1 M disodiumhydrogenphosphate dihydrate solution (170 mL,pH adjusted to 11.35 with 1 M NaOH) at 15° C. The pH of the solution wasre-adjusted to 11.4 by the addition of 1 M NaOH. 3 mL ofN-hexadecanoylglutamic acid γ-N-hydroxysuccinimide ester (248.1 mg, 0.51mmol) dissolved in N-methyl-pyrrolidone containing 0.105% w/w 1 M H₂SO₄was added dropwise during 8 min.

[0108] Samples were drawn out during 21.5 hours of reaction at 15° C.and added 0.1 M sodiumdi-hydrogenphosphate dihydrate solution containing1 mg/mL glycine, pH 7.5. The final reaction mixture was diluted withwater (300 mL) and the pH was adjusted to 8.0 by addition of glacialacid.

[0109] Yield: By analytical RP-HPLC the reaction mixture was shown tocontain 78-80% (by area) ofArg³⁴Lys²⁶-[N-ε-(γ-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷ and 0.24-1.67% (byarea) of Arg³⁴Lys²⁶-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-17. TABLE 2 Thecontent of Arg³⁴Lys²⁶-[N-ε-(γ-Glu(N- hexadecanoyl))]-GLP-1⁷⁻³⁷ (γ-Glu)and Arg³⁴Lys²⁶-[N- ε-(α-Glu(N-hexadecanoyl))]-GLP-1⁷⁻³⁷ (α-Glu) vs. timeof reaction. (Data from Example 8). Time γ-Glu α-Glu (hrs) (% by area)(% by area) 0.17 78.01 0.24 1 79.35 0.31 2 79.45 0.36 4 80.49 0.45 21.580.27 1.67

1. A method for producing an N-acylated peptide, said method comprising:a) reacting a peptide having at least one free amino group with anacylating agent of the general formula I

 wherein n is 0-8; R¹ is COOR⁴; R² is a lipophilic moiety; R³ togetherwith the carboxyl group to which R³ is attached designate a reactiveester or a reactive N-hydroxy imide ester; and R⁴ is selected fromhydrogen, c₁₋₁₂-alkyl and benzyl, under basic conditions in an aqueousmixture containing less than 10% w/w aprotic polar solvent; and b) if r⁴in the acylating agent of step a) is not hydrogen, saponifying theacylated peptide ester group (COOR⁴) under basic conditions; in order toproduce said n-acylated peptide.
 2. The method according to claim 1,wherein said reaction in step a) takes place in an aqueous mixturecontaining less than 8% w/w aprotic polar solvent.
 3. The methodaccording to claim 1, wherein said reaction in step a) takes place in anaqueous mixture containing less than 5% w/w aprotic polar solvent. 4.The method according to claim 1, wherein said reaction in step a) takesplace in an aqueous mixture containing less than 3% w/w aprotic polarsolvent.
 5. The method according to claim 1, wherein the acylating agentis added to the reaction mixture in step a) as a solid.
 6. The methodaccording to claim 1, wherein said reaction in step a) takes place inthe presence of an aprotic polar solvent.
 7. The method according toclaim 6, wherein said aprotic polar solvent is selected from the groupconsisting of N-methyl-2-pyrrolidone, tetrahydrofurane anddimethylsulfoxide.
 8. The method according to claim 6, wherein all ofthe aprotic solvent is added to the reaction mixture as a solvent forthe acylating agent.
 9. The method according to claim 6, wherein theacylating agent is added to the reaction mixture as a solution which isstabilized by adding an acid.
 10. The method according to claim 9,wherein said acid is added to the aprotic polar solvent in aconcentration from 0.01% w/w to 1% w/w.
 11. The method according toclaim 9, wherein said acid is added to the aprotic polar solvent in aconcentration from 0.05% w/w to 0.5% w/w.
 12. The method according toany one of claims 9, wherein said acid is selected from the groupconsisting of sulphuric acid, methanesulphonic acid and trifluoroaceticacid.
 13. The method according to claim 1, wherein the reaction in stepa) takes place in the absence of an aprotic polar solvent.
 14. Themethod according to claim 1, wherein R⁴ is hydrogen.
 15. The methodaccording to claim 1, wherein R⁴ is selected from C₁-alkyl and benzyl.16. The method according to claim 1, wherein R³ together with thecarboxyl group to which R³ is attached designate a reactive N-hydroxyimide ester.
 17. The method according to claim 1, wherein the acylatedpeptide ester is saponified in step b) at a pH value in the range of10-14.
 18. The method according to claim 1, wherein the acylated peptideester is saponified in step b) at pH range from 9-13.
 19. The methodaccording to claim 1, wherein pH of the reaction mixture in step a) isfrom pH 9to pH
 13. 20. The method according to claim 1, wherein pH ofthe reaction mixture in step a) is from pH 10 to pH
 12. 21. The methodaccording to claim 1, wherein pH of the reaction mixture in step a) isfrom pH 11.0 to pH 11.5.
 22. The method according to claim 1, whereinthe temperature of the reaction mixture in step a) is in the range of0-50° C.
 23. The method according to claim 1, wherein the temperature ofthe reaction mixture in step a) is in the range from 5-40° C.
 24. Themethod according to claim 1, wherein the temperature of the reactionmixture in step a) is in the range from 10-30° C.
 25. The methodaccording to claim 1, wherein R² is selected from C₃₋₃₉-alkyl,C₃₋₃₉-alkenyl, C₃₋₃₉-alkadienyl and steroidal residues.
 26. The methodaccording to claim 25, wherein R²—C(═O)— is selected from the groupconsisting of lithocholoyl and hexadecanoyl.
 27. The method according toclaim 1, wherein said peptide used as starting material for step a) hasa peptide purity of at least 80 as determined by RP-HPLC.
 28. The methodaccording to claim 1, wherein said peptide used as starting material forstep a) has a peptide purity of at least 90% as determined by RP-HPLC.29. The method according to claim 1, wherein said peptide used asstarting material for step a) has a peptide purity of at least 93% asdetermined by RP-HPLC.
 30. The method according to claim 1, wherein saidpeptide used as starting material for step a) has a peptide purity of atleast 95% as determined by RP-HPLC.
 31. The method according to claim 1,wherein said peptide used as starting material for step a) has a peptidepurity of at least 97% as determined by RP-HPLC.
 32. The methodaccording to claim 1, wherein said peptide is selected from the groupconsisting of GLP-1, exendin-4, GLP-2, glucagon, insulin, analoguesthereof and derivatives of any of the foregoing.
 33. The methodaccording to claim 1, wherein said peptide is a GLP-1 agonist.
 34. Themethod according to claim 1, wherein said peptide is selected from thegroup consisting of exendin-3, exendin-4, Arg³⁴-GLP-1(7-37),Gly⁸-GLP-1(7-36)-amide, Gly⁸-GLP-1(7-37), Val⁸-GLP-1(7-36)-amide,Val⁸-GLP-1(7-37), Val⁸Asp²²-GLP-1(7-36)-amide, Val⁸Asp²²-GLP-1(7-37),Val⁸Glu²²-GLP-1(7-36)-amide, Val⁸Glu²²-GLP-1(7-37),Val⁸Lys²²-GLP-1(7-36)-amide, Val⁸Lys²²-GLP-1(7-37),Val⁸Arg²²-GLP-1(7-36)-amide, Val⁸Arg²²-GLP-1(7-37),Val⁸His²²-GLP-1(7-36)-amide, Val⁸His²²-GLP-1(7-37), des(B30) humaninsulin and analogues thereof.
 35. The method according to claim 1,wherein said peptide is selected fromHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2 (ZP-10) and analoguesthereof.
 36. The method according to claim 1, wherein the reactionmixture in step a) comprises a buffer which is suitable for maintaininga substantially constant pH during the reaction.
 37. The methodaccording to claim 1, wherein said peptide is not insulin or an analoguethereof.